All eukaryotic cells are regulated by phosphoinositide (PI) signal transduction pathways. In the PI cycle, PI is phosphorylated to precursors of many PI-derived second messengers and directly to polyphosphoinositide messengers that regulate essential cellular processes. This laboratory has biochemically characterized and isolated cDNAs encoding two novel families of phosphoinositide kinases. These enzymes synthesize both phosphatidylinositol 4,5-bisphosphate (PI4,5P2) and the apoptosis inhibiting and mitogenic 3-polyphosphoinositides, PI3,4P2 and PI3,4,5P3. One enzyme, PIPKII-beta, is associated with the P55 tumor necrosis factor (TNF-alpha) receptor (TNFR1) and is activated by this receptor. The proposed studies will investigate the cellular functions of this receptor-kinase interaction and study the structure and function of this novel kinase. This work will involve the following Specific Aims: (1) Investigate the structure and function of PIPKII-beta in phosphoinositide messenger generation. Study the function of conserved sequences with the objective of characterizing ATP binding, the catalytic core, PIP binding sites, and requirements for, and consequences of, oligomer formation. (2) Investigate TNFR1 signaling through PIPKII-beta. Determine which TNF-regulated cellular events are mediated by PIPKII-beta. Determine which phosphoinositide messengers are generated by TNF-alpha stimulation in vivo and PIPKII-beta's role. (3) Determine the mechanism by which TNFR1 modulates PIPKII-beta signaling. TNFR1 functions by assembling a complex of protein mediators; with the yeast two-hybrid screen, the site of TNFR1 interaction with PIPKII-beta and whether the function or activity of PIPKII-beta is mediated by associated proteins will be determined. (4) In collaboration with Drs. James Hurley and Glenn Prestwich, the 3-D structure of PIPKII isoforms with bound substrate(s) will be determined. Crystals of PIPKII-beta have already been grown which defract well. Additional approaches will be used to grow PIPKII-beta crystals which can be used for high resolution structure determination with bound ATP and PI3P and/or PI4P. A role for PIP kinase in TNF-alpha signaling and potentially apoptosis regulation has many implications for signaling in general and proliferative diseases, immunology, and cancer, in specific.